PROJECT SUMMARY Every movement requires fine control of the strength and speed of muscle contractions. The nervous system achieves graded control of muscle by recruiting motor neuron (MN) subtypes with diverse functional properties, such as target muscle fiber type, electrophysiology, or role in behavior. The functional diversity of MNs is vital to locomotion, but we still lack a basic understanding of how it is created. During development, the identity of MN subtypes is determined by transcriptional codes. We know much about how anatomical MN subtypes defined by target muscle are specified, but the molecular logic by which functional MN subtypes develop is practically unknown. Though anatomical MN subtypes are well segregated in tetrapods, functional subtypes are heavily intermingled complicating their study. A model system where functional subtypes of MNs are segregated and easily identifiable, like in the zebrafish, provides a unique opportunity to dissect their molecular determinants. The zebrafish has three main functional subtypes of MNs responsible for slow, intermediate, and fast swimming. These functional subtypes can be identified based on their soma position, muscle fiber type, birthdate, recruitment order during swimming, and intrinsic properties. Through preliminary analysis, I have found two transcription factors, Evi1 and Prdm16, that are expressed in clustered subpopulations of post-mitotic axial MNs in the zebrafish spinal cord. Evi1 and Prdm16 are known to be important for cell fate and subtype specification in other cell types, but have not yet been studied in the spinal cord. Based on their expression pattern and preliminary behavioral data of mutant animals, I hypothesize Evi1 and Prdm16 specify a functional subtype of MNs responsible for intermediate speed swimming. In Aim 1, I will determine the MN subtype Evi1 and Prdm16 mark using histological approaches and molecular analyses. In Aim 2, I will determine if Evi1 and Prdm16 specify subtype identity using loss-of-function and gain-of-function methods. This project will elucidate the roles of Evi1 and Prdm16 in MN specification and could reveal molecular mechanisms responsible for the determination of functional properties.